Arthritis, and particularly rheumatoid arthritis (RA), is a painful and often crippling disease that initially results in swollen and inflamed joints, but often progresses to deform or completely destroy joints. This is a result of the body mistakenly attacking its own cartilage. Cartilage is a specialized kind of connective tissue which is found in human adults in three forms: hyaline or glossy cartilage; elastic cartilage; and fibrocartilage. Hyaline cartilage is the type found in the ventral ends of ribs, in joints, and in the walls of the larger respiratory passages. It is the hyaline cartilage that provides a low friction surface to prevent bone from rubbing on bone during motion. As arthritis progresses, cartilage is damaged and bone may also start to erode. This results in severe pain and ultimate destruction of the joint itself.
Arthritis is a group of diseases affecting joints and the component tissues. Several types of arthritis are recognized, and these can be divided into several groups by their clinical course or pathological manifestations. The most common form of arthritis is Osteoarthritis (OA). Osteoarthritis is mainly caused by mechanical damage to the joints, either by repetitive use of particular joints as seen in athletes and physical laborers, or by overloading structural joints as seen in the knee joints of obese individuals.
The second most common form of the disease is RA, which is a chronic multisystem disease of unknown cause. RA is characterized by chronic inflammation of the synovium associated with considerable erosion of both cartilage and bone, particularly in and around the joints. RA is currently understood as an autoimmune disease in which the pathological process appears to start by the presentation of an unknown "rheumatoid" self-antigen by an antigen presenting cell. Studies addressing family history indicate a genetic predisposition wherein a particular amino acid sequence in the third hypervariable region of the HLA-DR molecule is a major genetic element conveying susceptibility to RA. See Lipsky P E, "Rheumatoid Arthritis" in Harrisons' Principles of Internal Medicine, 13th ed. McGraw-Hill, Inc., New York, N.Y.
The T cell receptor on CD4+ T cells, which form the target of the antigen, also plays an important role in the inflammatory process. The presentation of the antigen causes the activation of CD4+ T cells, with the consequent secretion of cytokines such as interleukin-2 (IL-2) and interferon-g (IFN-g). These cytokines induce clonal expansion of the T cells and activation of the cytokine network. These cytokines trigger the production of endothelial adhesion molecules (such as ICAM-1) whose expression in rheumatoid synovium enhances the activation of inflammatory cells in the joints. See Vitali C, Sciuto M, Bombardieri S., "Immunotherapy in Rheumatoid Arthritis: A Review", Int J Art Organs 1993:16;196-200.
The modern therapy for arthritic conditions begins with nonsteroidal anti-inflammatory drugs such as aspirin, anthranilic acid, and ibuprofen; and more aggressive therapies involve disease-modifying antirheumatic drugs, such as D-penicillamine, methotrexate, and sulfasalazine. However, these treatments are often deficient in their efficacy and tolerability, causing a wide range of serious side effects. More severe forms of the disease may even require surgery.
(A) Oral Tolerance
Novel therapies for treating arthritis include immune response modifiers, gene therapy, enzyme inhibitors, monoclonal antibodies and dietary therapy. Dietary therapy for arthritis has received a great deal of publicity over the years. Although scientific basis, at present, for dietary remedies is still in doubt, there are valid reasons for considering whether dietary management can successfully modify disease activity as we better understand its etiology and pathology. "Oral tolerance" is a long recognized method to induce peripheral immune response. It was first described by Wells in 1911 as a state in which systemic anaphylaxis in guinea pigs was prevented by previous feeding of hen's egg proteins. See Wells H, "Studies on the Chemistry of Anaphylaxis III. Experiments with Isolated Proteins, Especially Those of Hen's Eggs", J Infect Dis 1911:9:147-151.
Particularly, oral tolerance is thought to be an ideal candidate to consider as a treatment of RA because of the etiology of RA as an autoimmune disease. Orally administered autoantigens have shown activity in several experimental autoimmune models including experimental autoimmune encephalomyelitis, uveitis, myasthenia, diabetes, and collagen- and adjuvant-induced arthritis. See Weiner H L, Friedman A, Miller A, Khoury S J, Al-Sabbagh A, Santos L, Sayegh M, Nussenblatt R B, Trentham D E, Hafler D A "Oral Tolerance: Immunologic Mechanisms and Treatment of Animal and Human Organ-Specific Autoimmune Diseases by Oral Administration of Autoantigens", Annu Rev Immunol 1994; 12:809-837.
The mechanism of how oral tolerance works is, at this time, unclear. The primary mechanisms by which an orally administered antigen induces tolerance are believed to be via the generation of active suppression or clonal anergy.
Collagen-induced arthritis (CIA) in experimental animals is the best known animal model for human RA. See Durie F H, Fava R A, Noelle R J, "Collagen-Induced Arthritis as a Model of Rheumatoid Arthritis" See Clin Immu and Immupath 1994; 73:11-18 and Staines N A, Wooley P H, "Collagen Arthritis-What Can it Teach Us?" Brit J Rheum 1994;33:798-807. It was first described by Trentham in 1977, see Trentham D E, Townes A S, Kang A H "Autoimmunity to Type II Collagen: An Experimental Model of Arthritis", J Exp Med 1977;146:857-868, and has been demonstrated to resemble human RA sufficiently to now be recognized as an important experimental tool. It is generally induced in susceptible strains of experimental animals (such as mice and rats) by immunization with heterologous type-II collagen (CII) isolated from a heterologous species. See Courtenay J S, Dallman M J, Dayyan A D, Martin A, Mosedale B, "Immunization Against Heterologous Type II Collagen Induced Arthritis in Mice", Nature 1980;283-665. In a susceptible strain of mice (DBA/1), immunization with CII initiates a combined humoral and cellular immune response targeted to joint tissues, where the antigen is predominantly located. Differences between the animal model and the human RA include:
(1) the model is an induced state and therefore does not occur spontaneously, as in humans; PA1 (2) the model lacks many extra-articular manifestations of the human RA including subcutaneous nodules and pulmonary fibrosis; and PA1 (3) the induction of the disease is of rapid onset in the model, which is different from humans in that it typically takes years.
Nevertheless, CIA is the best available animal model for human RA.
Intragastric administration of soluble Type II collagen (CII) prior to immunization with CII has been shown to suppress the incidence of CIA in DBA/1 Lac J mice, and WA/KIR rats. See Nagler-Anderson C, Bober L A, Robinson M E, Siskind G W, Thorbecke G J, "Suppression of the Type II Collagen-Induced Arthritis by Intragastric Administration of Soluble Type II Collagen", Proc Natl Acad Sci USA 1986;83:7443-7446 and Thompson H S G, Harper N, Devan D, Staines N A, "Suppression of CIA by Oral Administration of Type II Collagen: Changes in Immune and Arthritic Responses Mediated by Active Peripheral Suppression" Autoimmunity 1993;16:189-199.
Adjuvant-induced arthritis in Lewis rats was also shown to be suppressed by oral administration of soluble CII. See Zhang Z J, Lee C S Y, Lider O, Weiner H, "Suppression of Adjuvant Arthritis in Lewis Rats by Oral Administration of Type II Collagen", J Immunol 1990;145:2489-2493. The type of immunogen as well as the type of toleragen seems to be very important in exerting their effect in inducing and protecting the animal.
Cartilage is produced by cells called chondrocytes which synthesize and deposit around themselves a matrix of macromolecules that are known as collagen and proteoglycans. A remarkable function of cartilage tissue is that it replenishes itself in response to mechanical forces placed upon it.
A number of collagen types have been identified which provide the tough connective character of cartilage. The proteoglycans consist mainly of the high molecular weight molecules known as glycosaminoglycans (GAG) which include hyaluronic acid and chondroitin sulfate. GAGs were previously known as mucopolysaccharides. An overview of GAG's and their application in OA therapy is presented by Paroli, et al., in "A Pharmacological Approach to Glycosaminoglycans" Drugs Expth. Clin. Res. VII (1) 9-20 (1991).
A review of the various types of collagen can be found in Protein Profile, Vol. 1, 1994, pages 550-571; P. Sheterline, Ed. A review of the capacity of the immune system to discriminate between self and non-self structures and a discussion of how the immune system normally interacts with cartilage and how such interactions can lead to arthritis is set forth by Holmdahl, et al., in an article entitled "Autoimmune Recognition of Cartilage Collagens", Annals of Medicine 25:251-264, 1993. Type II collagen may be the best known oral toleragen for arthritis, however, there may exist other potential toleragens derived from non-cartilaginous tissues, such as vitreous humor, neural tubes and neural retina.
U.S. Pat. No. 5,529,786 to Moore discloses the use of animal tissue containing a therapeutic amount of CII for the treatment of RA in humans. This patent describes the animal tissue as preferably being chicken cartilage obtained from chickens less than about one year of age. Other animal tissues disclosed are bovine cartilage, the vitreous humor of eyes and a variety of other animals.
U.S. Pat. No. 4,473,551 to Schinitsky discloses a composition for the treatment of inflammatory disorders (such as RA, OA, acne, psoriasis and the like) which comprises animal cartilage and glucosamine. This patent describes the synergistic effect of a glucosamine and cartilages from whatever source derived, including shark and other marine dwellers, cattle, hogs, chicken and the like.
U.S. Pat. No. 5,075,112 to Lane discloses the use of finely divided shark cartilage for inhibiting tumor growth, arthritis, in particular RA, and inflammatory diseases with a vascular component. This patent does not suggest nor equate shark cartilage with any other mammal or avian cartilage.
U.S. Pat. No. 5,399,347 to Trentham, et al., discloses the use of a highly purified component from cartilage, whole CII protein, for the treatment of RA.
EP0254289B1 by Koepff, et al., discloses the treatment of arthritis through the administration of enzymatically hydrolyzed collagen from animal skins, animal bones, refined connective tissue or gelatin (Type I collagen) having an average molecular weight of from 10 to 80 KD.
Arthritis affects an estimated 40,000,000 people (15% of the population) in the United States. With an increasing survival time in the population, arthritis constitutes one of the greatest medical, social and economical problems in existence. The present invention furthers the state of the art of arthritis treatments and offers several advantages to presently accepted therapies.
(B) Notochord
As will be discussed and demonstrated below, notochord is a unique tissue to primitive groups of Osteichthyes, such as sturgeon and lamprey. Notochord appears in the post-gastrulation embryo as a very specialized mesoderm. In vertebrates, notochord serves as a core around which mesodermal cells gather to form the vertebrae (i.e., the notochord is the precursor of the vertebral column), but it disappears by the end of the embryonic stage. In the most primitive chordates, however, the notochord is retained as a primitive substitute for a vertebral column. Sturgeon and lamprey maintain a significant amount of notochord tissue in their vertebral column even in the adult stage. Regular mesoderm gives rise to the connective tissues of the body, such as hyaline cartilage and CII within. In fact, sturgeon notochord collagen may be a precursor form of CII, but it certainly is not CII. Notochord and cartilage are evolutionarily, developmentally, functionally, and anatomically different from each other. These differences in characteristics will be demonstrated below. The present invention relates to the use of intact and whole sturgeon notochord as well as its structural and chemical components (i.e., collagen derived from the notochord) for the treatment of RA and OA.
Support for the Applicant's position that notochord is different from cartilage can be found in an article by Miller and Mathews entitled, "Characterization of Notochord Collagen", published in Biochemical and Biophysical Research Communications, Vol. 60, No.1, 1974 and in an article published by Mathews entitled, "Comparative Biochemistry of Chondroitin Sulphate-Proteins of Cartilage and Notochord", published in Biochem J., (1971), 125, 37-46. These publications discuss the characterization of sturgeon notochord by chromatographic properties, amino acid composition, carbohydrate content, cyanogen bromide cleavage products of the component a-chain, the molecular parameters of tryptic-chymotryptic hydrolysates of chondroitin sulfate-protein, and the fraction of chondroitin sulfate-protein in a caesium chloride density gradient. These analyses point out some of the chemical similarities, but more importantly, they evidence the distinct chemical and structural differences between cartilage and notochord.
The following Tables 1 and 2 are from the Mathews (1971) publication, supra, and highlight the differences of notochord collagen from ox cartilage and sturgeon cartilage.
TABLE 1 ______________________________________ Molecular Parameters of Tryptic-Chymotryptic Hydrolysate of Chondroitin Sulfate-Protein and Products after Various Treatments Molecular TREATMENT Tissue Parameter None NaOH Papain Pronase ______________________________________ Ox cartilage [h] 102 60 60 96 Mn 43000 21000 21800 -- Mv 56000 27000 27000 51000 Sturgeon cartilage [h] 215 135 130 200 Mn 50000 25400 23000 -- Mv 66000 35000 34000 61000 Sturgeon notochord [h] 38 25 23 33 Mn 10000 5900 7100 -- Mv 15000 8100 7400 12000 ______________________________________ Mn viscosity molecular weight, was estimated from relationship [h] = 3.1 .times. 10.sup.-2 Mv.sup.0.74 for chondroitin sulphate A and [h] = 5.8 .times. 10.sup.-2 M.sup.0.74 for chondroitin sulphate C. (Mathews, 1971).
TABLE 2 ______________________________________ Fractionation of Chondroitin Sulphate-Protein in a Caesium Chloride Density Gradient Protein in Chondroitin Chondroitin Sulphate-Protein Source Zone Protein* sulphate* from each Zone (%) ______________________________________ Ox cartilage A 10 0 -- B 20 &lt;5 -- C 70 &gt;95 10.4 Sturgeon cartilage A 10 0 -- B 10 &lt;4 -- C 80 &gt;96 7.0 Sturgeon Notochord A 40 6 69 B 40 30 30 C 20 64 21 ______________________________________ The volumes and average densities at 20.degree. C. of zones were: A, 3 ml 1.42; B, 5 ml, 1.47; C, 4 ml, 1.54. Protein in chondroitin sulphateprotei from each zone was calculated from amino acid analysis. (Mathews, 1971) *percentage of total